Traumatic spinal cord injury (SCI) is a damage to the spinal cord that results in loss or impaired motor and/or sensory function. SCI is a sudden and unexpected event characterized by high morbidity and mortality rate during both acute and chronic stages, and it can be devastating in human, social and economical terms. Despite significant progresses in the clinical management of SCI, there remain no effective treatments to improve neurological outcomes. Among experimental strategies, bioengineered scaffolds have the potential to support and guide injured axons contributing to neural repair. The major aim of this study was to investigate a novel composite type I collagen scaffold with micropatterned porosity in a rodent model of severe spinal cord injury. After segment resection of the thoracic spinal cord we implanted the scaffold in female Sprague-Dawley rats. Controls were injured without receiving implantation. Behavioral analysis of the locomotor performance was monitored up to 55 days postinjury. Two months after injury histopathological analysis were performed to evaluate the extent of scar and demyelination, the presence of connective tissue and axonal regrowth through the scaffold and to evaluate inflammatory cell infiltration at the injured site. We provided evidence that the new collagen scaffold was well integrated with the host tissue, slightly ameliorated locomotor function, and limited the robust recruitment of the inflammatory cells at the injury site during both the acute and chronic stage in spinal cord injured rats. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016.

Over the last few years different antibacterial technologies have been developed in order to obtain fabrics and fibers with antibacterial capabilities for use in hospital environments. High levels of sanitation are indeed required in order to reduce nosocomial cross-transmission of infections. Silver-coated fibers are particularly appealing for the production of antibacterial textiles, due to the outstanding properties of silver, characterized by a high degree of biocompatibility, an excellent resistance to sterilization conditions, and antibacterial properties with respect to different bacteria, associated with long-term efficiency. In this study an innovative patented low-cost technique to deposit silver on natural and synthetic substrates has been exploited to obtain silver-coated natural fibers (i.e. cotton and flax). Such natural fibers are largely used in the hospitals for the production of sheets, pillowcases and other textile products that should possess high levels of sanitation. The structure and morphology of the silver nanoclusters deposited onto natural fibers was observed by scanning electron microscopy (SEM), and the coating was quantitatively assessed by thermogravimetric analysis (TGA). Good silver coating stability resulted from several industrial washings performed on the samples. The antimicrobial capabilities of the treated fibers were confirmed by antibacterial tests with Escherichia Coli. Silver-coated natural fibers thus show potential for the development of antibacterial textiles with long-term efficiency that is particularly useful in healthcare settings.

Crosslinking and denaturation were two variables that deeply affected the performance of collagen-based scaffolds designed for tissue regeneration. If crosslinking enhances the mechanical properties and the enzymatic resistance of collagen, while masking or reducing the available cell binding sites, denaturation has very opposite effects, as it impairs the mechanical and the enzymatic stability of collagen, but increases the number of exposed cell adhesive domains. The quantification of both crosslinking and denaturation was thus fundamental to the design of collagen-based scaffolds for selected applications. The aim of this work was to investigate the extents of crosslinking and denaturation of collagen-based films upon dehydrothermal (DHT) treatment, that is, one of the most commonly employed methods for zero-length crosslinking that shows the unique ability to induce partial denaturation. Swelling measurements, differential scanning calorimetry, Fourier transform infrared spectroscopy, colorimetric assays for the quantification of primary amines, and mechanical tests were performed to analyze the effect of the DHT temperature on crosslinking and denaturation. In particular, chemically effective and elastically effective crosslink densities were evaluated. Both crosslinking and denaturation were found to increase with the DHT temperature, although according to different trends. The results also showed that DHT treatments performed at temperatures up to 120°C maintained the extent of denaturation under 25%. Coupling a mild DHT treatment with further crosslinking may thus be very useful not only to modulate the crosslink density, but also to induce a limited amount of denaturation, which shows potential to partially compensate the loss of cell binding sites caused by crosslinking. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 186-194, 2016.

Autologous nerve grafting is the current gold standard treatment for peripheral nerve injury, in cases where direct suturing of nerve ends is not possible. Even though the functional restoration achieved by the autograft is not optimal, autologous nerve tissues still show higher regenerative capability than several synthetic conduits available in the clinical setting, the latter used only for gaps that do not exceed 3 cm in length. The aim of this chapter is to highlight how bio-mimicry, inspired by nerve development, structure and spontaneous regeneration following mild nerve injury, can help in the design of synthetic templates with optimized bioactivity for nerve regeneration.

A porous collagen-based hydrogel scaffold was prepared in the presence of iron oxide nanoparticles (NPs) and was characterized by means of infrared spectroscopy and scanning electron microscopy. The hybrid scaffold was then loaded with fluorescein sodium salt as a model compound. The release of the hydrosoluble species was triggered and accurately controlled by the application of an external magnetic field, as monitored by fluorescence spectroscopy. The biocompatibility of the proposed matrix was also tested by the MTT assay performed on 3T3 cells. Cell viability was only slightly reduced when the cells were incubated in the presence of the collagen-NP hydrogel, compared to controls. The economicity of the chemical protocol used to obtain the paramagnetic scaffolds as well as their biocompatibility and the safety of the external trigger needed to induce the drug release suggest the proposed collagen paramagnetic matrices for a number of applications including tissue engeneering and drug delivery.

The microstructural, mechanical, compositional, and degradative properties of a nerve conduit are known to strongly affect the regenerative process of the injured peripheral nerve. Starting from the fabrication of micropatterned collagen-based nerve guides, according to a spin-casting process reported in the literature, this study further investigates the possibility to modulate the degradation rate of the scaffolds over a wide time frame, in an attempt to match different rates of nerve regeneration that might be encountered in vivo. To this aim, three different crosslinking methods, that is, dehydrothermal (DHT), carbodiimide-based (EDAC), and glutaraldehyde-based (GTA) crosslinking, were selected. The elastically effective degree of crosslinking, attained by each method and evaluated according to the classical rubber elasticity theory, was found to significantly tune the in vitro half-life (t1/2) of the matrices, with an exponential dependence of the latter on the crosslink density. The high crosslinking efficacy of EDAC and GTA treatments, respectively threefold and fourfold when compared to the one attained by DHT, led to a sharp increase of the corresponding in vitro half-lives (ca., 10, 172, and 690 h, for DHT, EDAC, and GTA treated matrices, respectively). As shown by cell viability assays, the cytocompatibility of both DHT and EDAC treatments, as opposed to the toxicity of GTA, suggests that such methods are suitable to crosslink collagen-based scaffolds conceived for clinical use. In particular, nerve guides with expected high residence times in vivo might be produced by finely controlling the biocompatible reaction(s) adopted for crosslinking.

In the last decade cellulose-based hydrogels have been receiving increasing attention for a number of applications, due to their smart swelling behaviour, biodegradability and biocompatibility. Given the dramatic spreading of obesity and overweight in the industrialized countries and the lack of scientific consensus over currently available dietary supplements, it was recently proposed that such hydrogels might be used as orally administered bulking agents in hypocaloric diets, since the hydrogel swelling in the stomach may greatly reduce the space available for food intake, thus giving a sense of fullness. This study focused on the synthesis of cellulose-based hydrogels, starting from pharmaceutical and food grade cellulose derivatives, and showed that such hydrogels possess good swelling properties in water solutions mimicking the environmental conditions of the stomach and the intestine, as well as a good biocompatibility. The crosslinking agent used was a ‘zero-length’ crosslinker, i.e. a water soluble carbodiimide, which is washed out from the gel after the synthesis and does not affect the gel compatibility, as shown by preliminary biocompatibility assays. The experimental results confirmed that cellulosebased hydrogels might be a scientifically valid dietary adjuvant in the treatment of obesity and overweight, and provide further scientific evidence for future experiments on humans.

Purpose: The objective of this work was to develop composite hydrogels based on poly(ethylene glycol) diacrylate (PEGDA) and collagen (Coll), potentially useful for biomedical applications. Methods: Semi-interpenetrating polymer networks (semi-IPNs) were obtained by photo-stabilizing aqueous solutions of PEGDA and acrylic acid (AA), in the presence of collagen. Further grafting of the collagen macromolecules to the PEGDA/poly(AA) network was achieved by means of a carbodiimide-mediated crosslinking reaction. The resulting hydrogels were characterized in terms of swelling capability, collagen content and mechanical properties. Results and conclusions: The grafting procedure was found to significantly improve the mechanical stability of the IPN hydrogels, due to the establishment of covalent bonding between the PEGDA/poly(AA) and the collagen networks. The suitability of the composite hydrogels to be processed by means of stereolithography (SLA) was also investigated, toward creating biomimetic constructs with complex shapes, which might be useful either as platforms for tissue engineering applications or as tissue mimicking phantoms.

Collagen is one of the most used materials in scaffolding production; this is due to its peculiar characteristics that make the polymer highly biocompatible and efficient in regeneration induction and growth cone guidance. We aimed to investigate whether collagen could per se induce Schwann cell differentiation/proliferation and how it would do so. Results obtained in immortalized rat Schwann cells showed differential effects on several proliferation and differentiation markers depending on the type of collagen used to produce the scaffolds.

Dehydrothermal (DHT) crosslinking is routinely performed to increase the stiffness and the enzymatic resistance of collagen-based devices. Amide and ester bonds are formed among the collagen macromolecules, as a result of the high temperatures and high vacuum involved in the process. The extent of crosslinking is known to increase with the DHT temperature and duration, but simultaneous collagen denaturation might be induced. The aim of this work was to investigate the extent of crosslinking and denaturation of DHT-treated collagen-based films, by means of thermal and physicochemical analyses. With the ultimate goal of optimizing the DHT process, five different temperatures (110, 120, 140, 160 and 180°C) were used, while the DHT duration was kept constant (24 hours). Differential scanning calorimetry (DSC) was carried out to measure the denaturation temperature (Td) and enthalpy (ΔHd) of the collagen films. The reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBS) with primary amines (-NH2) allowed determining the number of free -NH2 in the collagen films, whereas Fourier transform infrared spectroscopy (FTIR) was used to investigate the chemical modifications occurring upon DHT treatment. Higher degrees of crosslinking were attained for increasing DHT temperatures, as demonstrated by reduced number of free -NH2, lower absorbance of amide II band (1545 cm-1) and higher Td values. However, the sharp reduction of ΔHd detected for samples treated at 140, 160 and 180°C indicated a significant denaturation associated to crosslinking. The analysis of the absorbance band at 1236 cm-1 confirmed that collagen denaturation was particularly pronounced for DHT temperatures higher than 120°C, suggesting that, at those temperatures, denaturation might predominate over crosslinking. Further stress relaxation tensile tests and dynamic mechanical analysis (DMA) are currently being performed to measure the stiffness of DHT-treated samples and to estimate the elastically effective crosslink density, according to the rubber elasticity theory.

The aim of this work was to investigate the structural features of type I collagen isoforms and collagen-based films at atomic and molecular scales, in order to evaluate whether and to what extent different protocols of slurry synthesis may change the protein structure and the final properties of the developed scaffolds. Wide Angle X-ray Scattering data on raw materials demonstrated the preferential orientation of collagen molecules in equine tendon-derived collagens, while randomly oriented molecules were found in bovine skin collagens, together with a lower crystalline degree, analyzed by the assessment of FWHM (Full Width at Half Maximum), and a certain degree of salt contamination. WAXS and FT-IR (Fourier Transform Infrared) analyses on bovine collagen-based films, showed that mechanical homogenization of slurry in acidic solution was the treatment ensuring a high content of super-organization of collagen into triple helices and a high crystalline domain into the material. In vitro tests on rat Schwannoma cells showed that Schwann cell differentiation into myelinating cells was dependent on the specific collagen film being used, and was found to be stimulated in case of homogenization-treated samples. Finally DHT/EDC crosslinking treatment was shown to affect mechanical stiffness of films depending on collagen source and processing conditions.

In this work, an innovative cellulose-based superabsorbent polymer (SAP) was experimentally assessed as an environmentally friendly alternative to acrylate-based SAPs, for the optimization of water consumption in agriculture. The cellulose-based SAP was synthesized and tested for its swelling capability in different aqueous media. The effectiveness of the SAP in agricultural applications was then evaluated by analyzing its performance after several absorption/desorption cycles, over a period of approximately 80 days, upon addition to different types of soil, i.e., white and red soil, for the cultivation of two varieties of plants typical of the Mediterranean area (tomatoes and chicory). The results confirmed that SAP-amended soil can store a considerable amount of water and can release it gradually to the plant roots when needed. The adoption of the proposed SAP in cultivations could thus represent a promising solution for the rationalization of water resources, especially in desert areas.

Poly(ethylene glycol) diacrylate (PEGDA) cryogels, particularly useful for biotechnological applications, are currently fabricated exploiting crosslinking systems that require long freezing/crosslinking times (20 h or longer). The aim of this work was to assess whether fast UV irradiation (up to 60 s) of frozen PEGDA solutions could be an advantageous alternative for cryogel production. By using different polymer concentrations and UV times, cryogels with highly interconnected macropores (about 50–90 μm) were produced. A gelation yield in the range 60–80% was recorded, with higher values obtained for low PEGDA concentrations (5 and 10% w/v). Interestingly, while decreasing the swelling and increasing the stiffness of the cryogels, a higher polymer concentration was also found to reduce the pore size. Furthermore, increasing the UV time resulted in significantly higher swelling and larger pores for 10% PEGDA samples, while having negligible effect on other cryogel types and/or features. Although deserving further exploration, fast UV irradiation is an effective method to produce PEGDA cryogels with tunable properties.

In tissue engineering field, the production of a porous resorbable matrix, termed scaffold, allows to host cells and guide them towards the synthesis of physiological tissue. Porous scaffolds provide mechanical stability and an initial framework for migrating cells and vascular infiltration. Sustained delivery of bioactive molecules at the defect site may be also particularly important for tissue regeneration. In this context, the goal of this work was the fabrication of highly porous collagen-based scaffolds incorporating uniformly dispersed poly(lactide-co-glycolide) (PLGA) microparticles as depots for the sustained and localized delivery of bioactive molecules. Collagen scaffolds loaded with different amounts of PLGA-microparticles were prepared by freeze-drying and crosslinking. The scaffolds microstructure was assessed to evaluate the spatial distribution of microparticles and the achieved pore size. The impact of the microparticles on the scaffolds stiffness was investigated through compression tests. Preliminarily, the cell-microparticles interactions were also evaluated by imaging of cell morphology in vitro, adopting a human derived epithelial cell model. The experimental findings showed that collagen scaffolds with different amounts of uniformly dispersed PLGA-microparticles were successfully produced. The microparticles did not negatively affect the scaffold porous structure, while acting as a mechanical reinforcement. Additionally, microparticles show high permissiveness to cell adhesion, and the interactions between microparticles and epithelial cell membranes did not interfere with the correct cells morphological differentiation. Such promising results suggest the potential of the developed scaffolds for tissue engineering applications.

INTRODUCTION Peripheral nerve injuries often result in painful neuropathies owing to reduction in motor function and sensory perception. When large nerve gaps exist (20mm or longer in humans), sensory nerve autografts are conventionally used to treat neural defects. The main issues related to autografts are shortage of donor nerves, a mismatch of donor nerve size with the recipient site, and occurrences of neuroma formation. Recent advances in nanotechnology and tissue engineering have been found to cover a broad range of applications in regenerative medicine and offer the most effective strategy to repair neural defects. Prior work in this area has shown the utility of collagen-based scaffolds for the regeneration of nerve tissue. This work focuses on the fabrication of collagen scaffolds with two different pore sizes, with the aim of evaluating the effects of pore size on the migration of Schwann cell lines. EXPERIMENTAL METHODS Scaffold fabrication and crosslinking Porous cylindrical scaffolds (diameter=2mm, length=10mm) with aligned channels were fabricated by freeze-drying a 2wt% collagen suspension along a one-dimensional temperature gradient (along the length of the cylindrical scaffold). Scaffolds with two different pore sizes were fabricated by freezing the collagen suspension at two different final freezing temperatures (-20°C and -60°C). The scaffolds were then subjected to dehydrothermal (DHT) cross-linking, followed by a carbodiimide based chemical crosslinking. Qualitative characterization of the pore structure was performed by means of scanning electron microscopy (SEM). Cell culture and cytocompatibility A rat Schwann cell line, RSC96, was expanded in monolayer culture in a 96-well plate. The plate was then incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, sterilized scaffolds were placed vertically to the wells of the 96-well plate and incubated again at 37°C and 5% CO2. At 1, 3, 7, and 10 days, the cell-seeded scaffolds were fixed in 10% formalin and processed for paraffin embedding. Schwann cells were quantified by embedding the cell-seeded scaffolds in paraffin blocks, sectioning, staining them with hematoxylin & eosin stain (H&E stain) and visualizing under a microscope. MTT assay was also performed at 1, 3, 7 and 10 days to evaluate the cell viability. RESULTS AND DISCUSSION SEM demonstrated that both freezing temperature and rate of freezing affect significantly the pore size. As shown in Fig.1, lower temperatures (-60°C) resulted in smaller pore sizes (~85µm), while higher temperatures (-20°C) resulted in much larger pores (~120µm). The longitudinal sections of the samples showed that the pores were in axial orientation disregard of the freezing temperature. MTT assay revealed that cell viability on the two different types of scaffolds increased gradually from first to the tenth day after seeding. Although there was not much difference between the two porous scaffolds on day 1, 3 and 7, on day 10 there was a slight increase in the cell number in the scaffolds with a larger pore size (-20°C). In spite of the different pore dimensions under investigation, the cell migration studies revealed that Schwann cells could migrate through the entire length of both types of scaffolds, by day 7. CONCLUSION Both types of scaffolds were found to support Schwann cell growth and migration, which is the key factor required for the regeneration of nerve tissue. Further studies are proposed regarding the addition of laminin and the evaluation of its effects on the cell growth and migration. REFERENCES 1. W. Daly et al., J. R. Soc. Interface 9:202-221, 2012.

Tubular scaffolds demonstrated to be able to reconnect the proximal and distal stumps of transected peripheral nerves and induce regeneration of the lost nerve trunk. Recently, a spinning technique has been developed, able to produce tubular collagen-based scaffolds characterized by a radially patterned microporosity. The technique is based on the centrifugal sedimentation of collagen taking place when a cylinder, containing an aqueous collagen suspension, is rotated rapidly around its axis. In this work, the centrifugation process was modeled by means of the Lamm differential equation for collagen concentration, with the assumption that sedimentation and diffusion coefficients were dependent on the local concentration, according to appropriate scaling laws. With such assumptions, the model was able to predict the actual tube formation and its inner radius, in good agreement with the experimental results. The possibility to predict the final scaffold inner diameter as a function of the processing parameters has a fundamental importance for the set up of a precise fabrication method, which does not make use of any complex mold. This would significantly reduce the production complexity and the extent of scaffold manipulation during production, resulting in a cleaner production process and safety of the device.

Bioactive food-preserving materials are based on the use of a natural antimicrobial compound loaded in a carrier material, which is able to trigger its release when requested and to modulate the rate of release, thus using either toxic or inhibitory properties against pathogens or bacteria due to food decomposition. In this study, the Schiff base formation for chitosan functionalization was achieved by the reaction of chitosan with cinnamaldehyde at different concentrations. Cinnamaldehyde is an aromatic α,β-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. It has been shown to exert antimicrobial action against a large number of microorganisms including bacteria, yeasts, and mould. The formation of the Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier. The reaction kinetics was investigated by means of rheological measurements, while infrared spectroscopy was used to assess the efficacy of the functionalization. The addition of nanometric graphene stacks to the cinnamaldehyde-functionalized chitosan films was evaluated with the aim to increase the mechanical properties of the film. Finally, the films were tested for antifungal properties with bread slices against a selected mould line.

In this chapter, we aim at providing an up-to-date review on nerve tissue engineering, focusing on both the peripheral and the central nervous systems (PNS and CNS, respectively). After introducing the pathophysiology of nerves and the social impact of nerve injuries, we overview the therapeutic approaches oriented toward inducing nerve regeneration, involving cellular, molecular, and scaffold-based strategies. A section is dedicated specifically to the PNS, with a critical focus on the actual therapeutic potential of experimental devices for the development of tissue-engineered medical products. A case study regarding the implementation of micropatterned collagen-based conduits in a clinical trial on PNS regeneration is also presented. Another section is dedicated to the ongoing research investigating the regenerative mechanisms of the CNS. In this context, spinal cord injury is assumed as a model lesion, for which complex tissue-engineered devices are being developed, at least in animal studies. With such a structure, this chapter is intended to provide a comprehensive, though not exhaustive, overview of nerve tissue engineering, which might be useful to students, researchers, clinicians, and biomedical entrepreneurs.

The specific design of a collagen scaffold containing iron oxide nanostructures capped by a TiO2 (anatase) layer is reported. The TiO2 shell is proposed with a dual role: as an innovative and biocompatible cross-linker agent, providing binding sites to the protein moiety, through the well-known TiO2 chemical affinity towards carboxyl groups, and as a protective surface layer from oxidation for the paramagnetic core. Simultaneously, the presence of the nanostructures confers to the collagen gel the sensitivity to an external stimulus, i.e. the application of a magnetic field. The hybrid biomaterial was demonstrated to be healthy and was proposed as a smart scaffold for the on demand release of bioactive compounds. The tunable release upon magnetic field application of a model protein, i.e. myoglobin, was investigated. Myoglobin was loaded in the microporous material and the discharging was induced by consecutive magnet applications, obtaining the release of the protein with a high spatio-temporal and dosage control.

Several bioengineering approaches have been proposed for peripheral nervous system repair, with limited results and still open questions about the underlying molecular mechanisms. We assessed the biological processes that occur after the implantation of collagen scaffold with a peculiar porous microstructure of the wall in a rat sciatic nerve transection model compared to commercial collagen conduits and nerve crush injury using functional, histological and genome wide analyses. We demonstrated that within 60 days, our conduit had been completely substituted by a normal nerve. Gene expression analysis documented a precise sequential regulation of known genes involved in angiogenesis, Schwann cells/axons interactions and myelination, together with a selective modulation of key biological pathways for nerve morphogenesis induced by porous matrices. These data suggest that the scaffold's microstructure profoundly influences cell behaviors and creates an instructive micro-environment to enhance nerve morphogenesis that can be exploited to improve recovery and understand the molecular differences between repair and regeneration.

The aim of this work was to assess the diffusive properties of poly(ethylene glycol) diacrylate (PEGDA)-based hydrogels, derived from low MW prepolymers, in view of potential biomedical applications. Several hydrogels were synthesized through UV irradiation of PEGDA solutions for different exposure times. Swelling measurements in distilled water were performed to estimate the yielded crosslink density, while swelling tests at 37 °C in selected media allowed to analyze the mesh size changes induced by various pH and ionic strength (IonS) conditions. The transport of glucose and insulin through thin hydrogel membranes was finally assessed in a modified Ussing chamber at physiological values of pH and IonS (7.4 and 150 mM, respectively). Results showed that the swelling was dependent on the IonS (with swelling reductions up to 20–30% for IonS increases in the range 0–300 mM) and, to a lesser extent, on the pH of the surrounding medium (with swelling increments of about 10% for increasing pH in the range 2.5–11). All hydrogels were also permeable to glucose and insulin, which displayed comparable diffusion coefficients (in the order of 10−6 cm2/s). Specific interactions between glucose and the polymer chains were evidenced by values of the partition coefficient higher than unity. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 44380. © 2016 Wiley Periodicals, Inc.

The aim of this work was the superficial activation, by means of plasma treatments, of crosslinked collagen-based scaffolds for nerve regeneration, in order to immobilize anionic and cationic microcapsules (MCPs) for drug delivery. Matrices with axially oriented pores have the potential to improve the regeneration of peripheral nerves and spinal cord by physically supporting and guiding the growth of neural structures across the site of injury. To improve mechanical resistance and stability in water solutions, it is necessary to crosslink collagenous fibres by formation of amide bonds with consequent reduction of free amino and carboxylic groups useful for immobilization approach of drug delivery systems like MCPs. Plasma chemical processes represent a successful approach because allow polar groups to be grafted on the surface, without modifying the massive properties of the bulk. Plasma surface modification was performed in a capacitively-coupled rf (13.56 MHz) glass reactor fed with different precursors like N2, H2O, C2H4 to study the effect of plasma parameters on the chemical properties of the resulting material and its ability to improve the immobilization of polyelectrolyte MCPs. Cylindrical scaffolds were synthesized by freeze-drying technique and dehydrothermally crosslinked. Polyelectrolyte capsules were obtained by LbL method. Scaffolds were characterized by means of WCA and XPS. Fluorescence microscopy was used to verify MCPs immobilization. After treatments, scaffolds became hydrophilic and able to absorb water. The success of grafting, on the external surface and within the scaffold core, was clearly revealed. The obtained results demonstrate that plasma processing of cross-linked collagen allows to enhance MCPs immobilization and that, by changing the typology of functional groups on the plasma treated surfaces, a different attitude to immobilize negatively or positively charged MCPs is observed.

Wound closure represents a primary goal in the treatment of very deep and/or large wounds, for which the mortality rate is particularly high. However, the spontaneous healing of adult skin eventually results in the formation of epithelialized scar and scar contracture (repair), which might distort the tissues and cause lifelong deformities and disabilities. This clinical evidence suggests that wound closure attained by means of skin regeneration, instead of repair, should be the true goal of burn wound management. The traditional concept of temporary wound dressings, able to stimulate skin healing by repair, is thus being increasingly replaced by the idea of temporary scaffolds, or regenerative templates, able to promote healing by regeneration. As wound dressings, polymeric hydrogels provide an ideal moisture environment for healing while protecting the wound, with the additional advantage of being comfortable to the patient, due to their cooling effect and non-adhesiveness to the wound tissue. More importantly, recent advances in regenerative medicine demonstrate that bioactive hydrogels can be properly designed to induce at least partial skin regeneration in vivo. The aim of this review is to provide a concise insight on the key properties of hydrogels for skin healing and regeneration, particularly highlighting the emerging role of hydrogels as next generation skin substitutes for the treatment of full-thickness burns.

The present work deals with the development of a biodegradable superabsorbent hydrogel, based on cellulose derivatives, for the optimization of water resources in agriculture, horticulture and, more in general, for instilling a wiser and savvier approach to water consumption. The sorption capability of the proposed hydrogel was firstly assessed, with specific regard to two variables that might play a key role in the soil environment, that is, ionic strength and pH. Moreover, a preliminary evaluation of the hydrogel potential as water reservoir in agriculture was performed by using the hydrogel in experimental greenhouses, for the cultivation of tomatoes. The soil-water retention curve, in the presence of different hydrogel amounts, was also analysed. The preliminary results showed that the material allowed an efficient storage and sustained release of water to the soil and the plant roots. Although further investigations should be performed to completely characterize the interaction between the hydrogel and the soil, such findings suggest that the envisaged use of the hydrogel on a large scale might have a revolutionary impact on the optimization of water resources management in agriculture.

The aim of this study was to investigate the synthesis of chitosan nanoparticles for growth factor delivery in bone tissue engineering. Chitosan nanoparticles were synthesized via a modified precipitation process and their morphology and dimensions characterized by means of scanning electron microscopy (SEM) and dynamic light scattering (DLS), respectively. In particular, both chitosan molecular weight and concentration were varied during the synthesis to assess the effect of those variables on the particle size and morphology. The stability of the nanoparticles in aqueous media was also assessed, by measuring the average increase of the particle size with time. A specific particle formulation was then selected and loaded with a model molecule, i.e. an oligopeptide derived from the bone morphogenetic protein BMP2. The effect of the nanoparticles on the viability of osteoblast-like MG63 cells was finally assessed in a cytotoxicity assay. The encouraging results obtained in this study, although preliminary, suggested the possible use of chitosan nanoparticles for bone tissue engineering.

In this work, a mixture of a sodium salt of carboxymethylcellulose (CMCNa) and polyethylene glycol diacrylate (PEGDA700) was used for the prepara- tion of a microporous structure by using the combination of two different procedures. First, physical foaming was induced using Pluronic as a blowing agent, followed by a chemical stabilization. This second step was carried out by means of an azobis(2-methylpropionamidine)dihydrochloride as the thermoinitiator (TI). This reaction was activated by heating the sample homo- geneously using a microwave generator. Finally, the influence of different CMCNa and PEGDA700 ratios on the final properties of the foams was inves- tigated. The viscosity, water absorption capacity, elastic modulus and porous structure were evaluated for each sample. In addition, preliminary biological characterization was carried out with the aim to prove the biocompatibility of the resulting material. The foam, including 20% of PEGDA700 in the mixture, demonstrated higher viscosity and stability before thermo-polymerization. In addition, increased water absorption capacity, mechanical resistance and a more uniform microporous structure were obtained for this sample. In particu- lar, foam with 3% of CMCNa shows a hierarchical structure with open pores of different sizes. This morphology increased the properties of the foams. The full set of samples demonstrated an excellent biocompatibility profile with a good cell proliferation rate of more than 7 days.

Rapid prototyping techniques have been investigated for the production of biomedical devices that perfectly fit the patient's tissue defect (e.g. for bone and dental applications) and/or reproduce the microstructure of the tissue or organ of interest. The possibility to create patient-specific devices has been recently exploited for the creation of tissue engineer-ing scaffolds, i.e. porous, resorbable matrices, which stimulate cell functions and induce tissue regenera-tion by providing cells with appropriate physical, mechanical and biochemical cues. Poly(ethylene glycol) (PEG)-based hydrogels, although intrinsically non-biodegradable and non-bioactive, show great promise as tissue engineering scaffolds, due to their ability to be covalently linked to bioactive and/or degradable moieties, that elicit specific cell responses, and to their fast and biocompatible formation under ultra-violet (UV) exposure. In this work, poly(ethylene glycol)-based hydrogels, containing bioactive moieties, were photopolymerized and characterized in terms of mechanical, swelling and degradation properties. The production of hydrogels possessing a complex shape was finally investigated by means of stereolithography, a rapid prototyping technique which is able to build a three-dimensional object, starting from the CAD model, by guiding an ultravio-let laser beam on the surface of a photosensitive solution. The results demonstrated that the developed hydrogel formulations allow the creation of biomimetic constructs with complex shapes, which might be useful as platforms for tissue engineering or as tissue mimicking phantoms.

In this study we investigated the impact of three different sterilization methods, dry heat (DHS), ethylene oxide (EtO) and electron beam radiation (β), on the properties of cylindrical collagen scaffolds with longitudinally oriented pore channels, specifically designed for peripheral nerve regeneration. Scanning electron microscopy, mechanical testing, quantification of primary amines, differential scanning calorimetry and enzymatic degradation were performed to analyze possible structural and chemical changes induced by the sterilization. Moreover, in vitro proliferation and infiltration of the rat Schwann cell line RSC96 within the scaffolds was evaluated, up to 10 days of culture. No major differences in morphology and compressive stiffness were observed among scaffolds sterilized by the different methods, as all samples showed approximately the same structure and stiffness as the unsterilized control. Proliferation, infiltration, distribution and morphology of RSC96 cells within the scaffolds were also comparable throughout the duration of the cell culture study, regardless of the sterilization treatment. However, we found a slight increase of chemical crosslinking upon sterilization (EtO < DHS < β), together with an enhanced resistance to denaturation of the EtO treated scaffolds and a significantly accelerated enzymatic degradation of the β sterilized scaffolds. The results demonstrated that β irradiation impaired the scaffold properties to a greater extent, whereas EtO exposure appeared as the most suitable method for the sterilization of the proposed scaffolds.

Collagen, the major component of the extracellular matrix, key factor of tissue architecture, provides tensile strength, cell-matrix and matrix-matrix interactions. 2-D and 3-D collagen constructs are widely used as tissue scaffolds in a variety of biomedical applications. Here we synthetized scaffolds structured as thin films (30-50 μm in thickness) and we characterized them from a structural and functional point of view. Type I collagen isolated from bovine tendon (Sigma Aldrich) was suspended at 0.5% w/v in dilute hydrochloric acid (pH=3-3.2) by mixing at 15,000 rpm in an overhead blender, under proper refrigeration. After degassing via centrifugation, the slurry was cast in polystyrene molds and dried for at least 48 hours at room temperature, to obtain dry collagen films. In order to modulate their mechanical properties and degradation rate, the samples were subjected to two different crosslinking treatments, either dehydrothermal crosslinking (DHT) only, at 121°C for 24 hours, or DHT treatment combined with chemical crosslinking by means of a water soluble carbodiimide (DHT/EDC). First, atomic force microscopy measurements of the films showed differences in structure reticulation. As expected, the DHT/EDC scaffold surface presented a more intricate fibrillar assembly, and a lower swelling degree after the first 24 hours (about 30% less). Once integrated into appositely fabricated polymeric devices, DHT and DHT/EDC scaffolds were tested in cellular oxygen consumption and proliferation assays. Fibroblasts, seeded at the same densities on both DHT and DHT/EDC substrates, displayed different oxygen consumption rate (OCR) within 48 hours, reflecting dissimilarities in terms of structural organization and oxygen diffusion efficiency. The obtained results could represent a useful approach to indicate some culture parameters, such as cell-seeding optimal values for the feasibility of tissue models, depending on scaffold structure design.

Le lesioni del midollo spinale nell’uomo sono molto eterogenee. L’incapacità di rigenerazione del midollo lesionato è attribuita all’instaurarsi di un ambiente inibitorio e alla formazione di una cicatrice gliale che funge da barriera chimica e meccanica alla rigenerazione assonale. L’impianto di scaffold microporosi rappresenta una valida strategia per guidare la rigenerazione, nel tentativo di ripristinare i collegamenti con i target di innervazione e promuovere il recupero funzionale. Porosità, distribuzione delle dimensioni dei pori, area superficiale specifica, interconnettività ed orientazione dei pori sono parametri cruciali che influenzano la bioattività dello scaffold. Lo scopo del presente lavoro è quello di modulare e caratterizzare la struttura microporosa di scaffold cilindrici in collagene, con porosità orientata in direzione longitudinale o assiale, destinati ad uno studio sulla rigenerazione del midollo spinale. Gli scaffold (3mm diametro, 3 cm lunghezza) sono stati realizzati mediante freezing unidirezionale di sospensioni di collagene di tipo I da derma bovino (a diverse concentrazioni), liofilizzazione e reticolazione termica e chimica. La porosità degli scaffold è stata quindi analizzata qualitativamente e quantitativamente mediante microscopia elettronica a scansione e ottica, al fine di determinare morfologia, omogeneità, diametro medio e grado di orientazione dei pori. L’analisi delle sezioni trasversali e longitudinali degli scaffold ha mostrato rispettivamente una distribuzione pressoché omogenea e una buona orientazione uniassiale dei pori per tutte le tipologie di campioni analizzate, evidenziando una leggera diminuzione della dimensione media all’aumentare della concentrazione di collagene utilizzata in fase di sintesi. Tuttavia, si è anche osservato un gradiente crescente del diametro medio dei pori lungo l’asse longitudinale degli scaffold, legato al gradiente di temperatura che si instaura durante il processo di freezing uniassiale. Inoltre, i trattamenti di reticolazione investigati sembrano non influenzare significativamente la microstruttura. Studi futuri saranno rivolti a comprendere l’effetto della microstruttura sul comportamento di cellule neuronali, immortalizzate e primarie, in vitro.

In the present work, we measured the degradation rate of different chitosan slurries. Several parameters were monitored such as temperature (25 °C, 37 °C, 50 °C); chitosan concentration (1% and 2% (w/V)); and polymer molecular weight. The samples were tested in dynamic sweep test mode. This test is able to provide a reliable estimation of viscosity variations of the slurries; in turn, these variations could be related to degradation rate of the system in the considered conditions. The resulting information is particularly important especially in applications in which there is a close relationship between physical properties and molecular structure.

Due to its intrinsic biocompatibility, degradability, and antibacterial properties, chitosan is widely explored for biomedical and pharmaceutical applications, especially for the development of tissue engineering scaffolds and controlled drug delivery systems. In this work, physically crosslinked chitosan-based particles with submicrometric size were synthesized by means of a modified coacervation process, starting from aqueous solutions differing for the chitosan molecular weight and concentration. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to analyse the particle morphology and the mean diameter yielded by the different synthesis parameters. Daily DLS measurements were also performed to monitor the expected swelling of the particles in a buffer solution, up to four days of storage. The experimental findings showed that submicrometric chitosan particles, with an average diameter in the range 150–400 nm, could be successfully produced, with both chitosan molecular weight and concentration affecting the particle size. Moreover, the smallest particles, among those synthesized, were found to be stable in water solutions up to three days. This seems to suggest the potential of the investigated particles for short-term biomedical applications, e.g., controlled drug delivery over time windows ranging from hours to days.

The aim of this work is to develop a novel approach to control the growth of food-borne and food-spoilage microorganisms while reducing the use of synthetic preservatives. Bioactive food-preserving systems are based on the use of a natural antimicrobial agent loaded in a carrier material, which is able to trigger its release once necessary and to control the rate of release, thereby exerting either lethal or inhibitory effects against food pathogens or spoilage microorganisms. In this study the Schiff base of chitosan was synthesized by the reaction with cinnamaldehyde at different concentrations (0,1%, 0,25%, 0,5% w/w of dry polymer). Cinnamaldehyde is an aromatic α,β-unsaturated aldehyde, and the major component in essential oils from some cinnamon species. It has been shown to exert antimicrobial activity against a wide range of microorganisms including bacteria, yeasts, and mould. The formation of the Schiff base is reversible under suitable conditions, and this might allow the release of the active cinnamaldehyde from chitosan, used as the carrier. The reaction kinetics was investigated by means of rheological analyses, while Fourier transform infrared spectroscopy (FTIR) was used to assess the efficacy of the functionalization. The results from FT-IR spectra highlighted the presence of the absorption peak of the Schiff base, which confirmed the reactivity of the nitrogen from amino group of chitosan and carbonyl carbon of the aldehyde to form imine. Moreover, the reaction rate was found to increase as higher percentages of cinnamaldehyde were used. Cinnamaldehyde-functionalized chitosan films were then prepared and tested for contact angle and antifungal properties in vitro. The envisaged application of the films for food packaging was also tested, by placing the films in direct contact with slices of bread. It was demonstrated that the cinnamaldehyde-functionalized chitosan films increased the shelf life of the product.

Porosity is a key parameter in the design of tissue engineering scaffolds, as bioactivity can be controlled and tailored to the synthesis of the target tissue by finely tuning the porous structure of the scaffolding biomaterial. This chapter discusses the effect of structural parameters, such as pore volume fraction, pore size and distribution, pore shape, pore interconnectivity and pore orientation, on the performance of sponge-like scaffolds, with a special focus on those directed to nerve regeneration.